K-ASSAY 

KAMIYA BIOMEDICAL COMPANY

KAMIYA BIOMEDICAL COMPANY

Mouse Serum Amyloid P
ELISA
For the quantitative determination of Serum Amyloid P (SAP)in mouse biological samples

Cat. No. KT-347

For Research Use Only.
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KAMIYA BIOMEDICAL COMPANYPRODUCT INFORMATION

Mouse Serum Amyloid P ELISA
Cat. No. KT-347
INTENDED USE
The Mouse Serum Amyloid P ELISA is a highly sensitive two-site enzyme-linked immunoassay (ELISA) for thequantitative determination of Serum Amyloid P (SAP) in mouse biological samples. For research use only.

INTRODUCTION
Mouse serum amyloid P (SAP) is a major acute phase protein that circulates as an ~230 kDa glycoprotein. It is
composed of 10 identical subunits arranged as two cyclic pentameric discs and contains ~10% carbohydrate. Basallevels of SAP may vary significantly in different inbred mouse strains but SAP is a major acute phase protein in all strains.
During acute chemical, physical or inflammatory stimulus, its concentration can increase 50-100 fold within 24-48 hours.
Changes can be readily detected in 4-6 hours. Measurement of the concentration of SAP is indicative of the extent andseverity of an inflammatory stimulus and can be used to assess various modalities of treatment.

PRINCIPLE
The principle of the double antibody sandwich ELISA is represented in Figure 1. In this assay the SAP present insamples reacts with the anti-SAP antibodies, which have been adsorbed to the surface of polystyrene microtiter wells.
After the removal of unbound proteins by washing, anti-SAP antibodies conjugated with horseradish peroxidase (HRP),
are added. This HRP-conjugated antibody forms a complex with the previously bound SAP. Following another washing
step, the enzyme bound to the immunosorbent is assayed by the addition of a chromogenic substrate, 3,3’,5,5’tetramethylbenzidine (TMB). The quantity of bound enzyme is proportional to the concentration of SAP in the sample
tested; thus, the absorbance, at 450 nm, is a measure of the concentration of SAP in the test sample. The quantity of
SAP in the test sample can be interpolated from the calibration curve constructed from the calibrators, and corrected forsample dilution.

Figure 1.
Anti-SAP Antibodies Bound To Solid Phase│
Calibrators and Samples Added│
SAP * Anti-SAP Complexes Formed│
Unbound Sample Proteins Removed│
Anti-SAP-HRP Conjugate Added│
Anti-SAP-HRP * SAP * Anti-SAP
Complexes Formed│
Unbound Anti-SAP-HRP Removed│
TMB Substrate Added│
Determine Bound Enzyme Activity

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KAMIYA BIOMEDICAL COMPANYCOMPONENTS
1. Diluent Concentrate
One bottle containing 50 mL of a 5X concentrated diluent running buffer.
2. Wash Solution Concentrate
One bottle containing 50 mL of a 20X concentrated wash solution.
3. Enzyme-Antibody Conjugate Concentrate
One vial containing 150 µL of a 100X concentrated affinity-purified anti-mouse SAP antibody conjugated with HRP in astabilizing buffer.
4. TMB Substrate Solution
One vial containing 12 mL of TMB and hydrogen peroxide in citric acid buffer at pH 3.3.
5. Stop Solution
One vial containing 12 mL of 0.3 M sulfuric acid. WARNING: Avoid contact with skin.
6. Microtiter Plate
Twelve removable eight-well strips in well holder frame. Wells are coated with affinity-purified anti-mouse SAP.
7. Mouse SAP Calibrator
One vial containing a lyophilized Mouse SAP Calibrator.
8. Positive Control
One vial containing 50 µL of serum with 0.1% sodium azide. See the Control Certificate for the concentration.

MATERIALS REQUIRED BUT NOT PROVIDED
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Test tubes
Precision pipettes (2 µL to 200 µL) for making and dispensing dilutions
Microplate washer/aspirator
Distilled or de-ionized H2O
Microplate reader
Assorted glassware for the preparation of reagents and buffer solutions
Timer

PRECAUTIONS
1. Read the instructions carefully before beginning the assay.
2. This kit is for research use only.
3. Great care has been taken to ensure the quality and reliability of this product. However, it is possible that in certain
cases, unusual results may be obtained due to high levels of interfering factors.
4. Preservatives
 Positive Control contains 0.1% sodium azide.
5. No additives or preservatives are necessary to maintain the integrity of the specimen. Avoid azide contamination.
6. Azide and thimerosal at concentrations higher than 0.1% inhibit the enzyme reaction.
7. Other precautions: Do not interchange kit components from different lots.
 Do not use kit components beyond the expiration date.
 Protect reagents from direct sunlight.
 Do not pipette by mouth.
 Do not eat, drink, smoke or apply cosmetics where reagents are used.
 Avoid all contact with the reagents by using gloves.
 Stop solution contains diluted sulfuric acid. Irritation to eyes and skin is possible. Flush with water after
contact.

REAGENT PREPARATION
1. Diluent Concentrate
The Diluent solution supplied is a 5X concentrate and must be diluted 1:5 with distilled or de-ionized water.

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2. Wash Solution Concentrate
The Wash Solution supplied is a 20X concentrate and must be diluted 1:20 with distilled or de-ionized water. Crystal
formation in the concentrate is not uncommon when storage temperatures are low. Warming of the concentrate to30-35°C before dilution can dissolve crystals.
3. Enzyme-Antibody Conjugate Concentrate
Calculate the required amount of working conjugate solution for each microtiter plate test strip by adding 10 µL
Enzyme-Antibody Conjugate to 990 µL of 1X Diluent for each test strip to be used for testing. Mix uniformly, but gently.
Avoid foaming.
4. TMB Substrate Solution
Ready to use as supplied.
5. Stop Solution
Ready to use as supplied.
6. Microtiter Plate
Ready to use as supplied. Unseal Microtiter Pouch and remove plate from pouch. Remove all strips and wells that will
not be used in the assay and place back in pouch and re-seal with desiccant.
7. Mouse SAP Calibrator
Add 1 mL of distilled or de-ionized water to the lyophilized Mouse SAP Calibrator and mix gently until dissolved. The
calibrator is now at a concentration of 3.4 µg/mL (the reconstituted calibrator should be aliquoted and frozen if future
use is intended). Prepare the Mouse SAP Calibrators immediately prior to use according to the table below. Mix well
between each step. Avoid foaming.
Calibrator
6
5
4
3
2
1
0

Concentration(ng/mL)
200
100
50
25
12.5
6.25
0

Calibrator Volume added
to 1X Diluent
40 µL Mouse SAP Calibrator
300 µL Calibrator 6
300 µL Calibrator 5
300 µL Calibrator 4
300 µL Calibrator 3
300 µL Calibrator 2

Volume of 1X Diluent
640 µL
300 µL
300 µL
300 µL
300 µL
300 µL
600 µL8. Positive Control
The concentration and recommended dilution are provided on the Control Certificate. Before use, briefly centrifuge thePositive Control to allow all of the liquid to collect in the bottom of the vial.

STORAGE AND STABILITY
1. Complete Kit
The expiration date for the kit is stated on the outer label. The recommended storage temperature is 4°C. Note: Seelong-term storage recommendations below for the Mouse SAP Calibrator and Positive Control.
2. Diluent
The 5X Diluent Concentrate is stable until the expiration date. The 1X working solution is stable for at least one weekfrom the date of preparation. Both solutions should be stored at 4°C.
3. Wash Solution
The 20X Wash Solution Concentrate is stable until the expiration date. The 1X working solution is stable for at least
one week from the date of preparation. Both solutions can be stored at room temperature (RT, 16-25°C) or at 4°C.
4. Enzyme-Antibody Conjugate
Undiluted anti-SAP-HRP conjugate should be stored at 4°C and diluted immediately prior to use.
conjugate solution is stable for up to 1 hour when stored in the dark.

The working

5. TMB Substrate Solution
The TMB Substrate Solution should be stored at 4°C and is stable until the expiration date.

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6. Stop Solution
The Stop Solution should be stored at 4°C and is stable until the expiration date.
7. Microtiter Plate
Anti-mouse SAP coated wells are stable until the expiration date and should be stored at 4°C in the sealed foil pouchwith a desiccant pack.
8. Mouse SAP Calibrator
The lyophilized Mouse SAP Calibrator should be stored at 4°C or frozen until reconstituted. The reconstituted calibrator
should be aliquoted and stored frozen. Avoid multiple freeze/thaw cycles. The working calibrator solutions should be
prepared immediately prior to use and are stable for up to 1 hour.
9. Positive Control
For storage longer than 7 days keep frozen until the expiration date. Storage less than 7 days can be at 4°C. Avoid
multiple freeze/thaw cycles.

INDICATIONS OF INSTABILITY
If the test is performing correctly, the results observed with the calibrator solutions should be within 20% of the expectedvalues.

SPECIMEN COLLECTION AND HANDLING
Blood should be collected by venipuncture and the serum separated from the cells, after clot formation, by centrifugation.
For plasma samples, blood should be collected into a container with an anticoagulant and then centrifuged. Care should
be taken to minimize hemolysis, excessive hemolysis can impact your results. Assay immediately or aliquot and store
samples at -20°C. Avoid repeated freezing/thawing.
For any sample that might contain pathogens, care must be taken to prevent contact with open wounds. No additives orpreservatives are necessary to maintain the integrity of the specimen. Avoid azide contamination.

ASSAY PROTOCOL
Dilution of Samples
The assay for quantification of SAP in samples requires that each test sample be diluted before use. For a single step
determination a dilution of 1:1,500 is appropriate for most serum/plasma samples. For absolute quantification of samples
that yield results outside the range of the calibration curve, a lesser or greater dilution might be required. If unsure ofsample level, a serial dilution with one or two representative samples before running the entire plate is highly
recommended.
To prepare a 1:1,500 dilution of sample, transfer 5 µL of sample to 495 µL of 1X Diluent. This gives you a 1:100 dilution.
Next, dilute the 1:100 sample by transferring 20 µL, to 280 µL of 1X Diluent. You now have a 1:1,500 dilution of yoursample. Mix thoroughly at each stage.

Procedure
1. Bring all reagents to RT before use.
2. Pipette 100 µL of
Calibrator 0 (0.0 ng/mL) in duplicate
Calibrator 1 (6.25 ng/mL) in duplicate
Calibrator 2 (12.5 ng/mL) in duplicate
Calibrator 3 (25 ng/mL) in duplicate
Calibrator 4 (50 ng/mL) in duplicate
Calibrator 5 (100 ng/mL) in duplicate
Calibrator 6 (200 ng/ml) in duplicate
3. Pipette 100 µL of diluted Positive Control (in duplicate) into pre designated wells.
4. Pipette 100 µL of diluted sample (in duplicate) into pre designated wells.

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5. Incubate the Microtiter Plate at 22°C (RT) for sixty (60 ± 2) minutes. Keep plate covered and level during incubation.
6. Following incubation, aspirate the contents of the wells.
7. Completely fill each well with appropriately diluted Wash Solution and aspirate. Repeat three times, for a total of four
washes. If washing manually: completely fill wells with diluted Wash Solution, invert the plate and pour/shake out thecontents in a waste container. Follow this by sharply striking the wells on absorbent paper to remove residual Wash
Solution. Repeat three times for a total of four washes.
8. Pipette 100 µL of appropriately diluted Enzyme-Antibody Conjugate to each well. Incubate at 22°C (RT) for sixty (60 ±2) minutes. Keep plate covered in the dark and level during incubation.
9. Wash and blot the wells as described in steps 6 and 7.
10. Pipette 100 µL of TMB Substrate Solution into each well.
11. Incubate at RT in the dark for precisely ten (10) minutes.
12. After ten minutes, add 100 µL of Stop Solution to each well.
13. Determine the absorbance at 450 nm of the contents of each well. Zero the plate reader to air
The absorbance of the final reaction mixture can be measured up to two hours after the addition of the Stop Solution.
However, good laboratory practice dictates that the measurement be made as soon as possible.

RESULTS
1. Subtract the average background value from the test values for each sample.
2. Using the results observed for the calibrators construct a calibration curve. The appropriate curve fit is that of a four –parameter logistics curve. A second-order polynomial (quadratic) or other curve fits may also be used.
3. Interpolate test sample values from the calibration curve.
concentration in original sample.

Correct for sample dilution factor to arrive at SAPQUALITY CONTROLIn accord with good laboratory practice, the assays for specific SAP require meticulous quality control. Each laboratory
should use routine quality control procedures to establish inter- and intra-assay precision and performance characteristics.

LIMITATION OF THE PROCEDURE
1. Reliable and reproducible results will be obtained when the assay procedure is carried out with a complete
understanding of the information contained in the package insert instructions and with adherence to good laboratory
practice.
2. Factors that might affect the performance of the assay include proper instrument function, cleanliness of glassware,
quality of distilled or de-ionized water, and accuracy of reagent and sample pipettings, washing technique, incubation
time or temperature.

FOR RESEARCH USE ONLY

KAMIYA BIOMEDICAL COMPANY
12779 Gateway Drive, Seattle, WA 98168
Tel: (206) 575-8068 Fax: (206) 575-8094
Email: LifeScience@k-assay.com
www.k-assay.com

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